The serine proteases of the intrinsic blood coagulation cascade art slowly neutralized by antithrombin (AT) (reviewed in (1)). This inhibition is secondary to the generation of 1:1 enzyme-AT complexes whose formation is dramatically enhanced by the mast cell product, heparin (2). Damus et al. (3) hypothesized that endothelial cell surface heparan sulfate proteoglycans (HSPGs) function in a similar fashion to accelerate coagulation enzyme inactivation by AT, and therefore are responsible for the non-thrombogenic properties of blood vessels. It was initially demonstrated that profusion of the hindlimbs of normal rodents and rodents deficient in mast cells with purified thrombin (T) and AT leads to a greatly elevated rate of T-AT complex formation and that the enzyme heparitinase as well as the natural heparin antagonist platelet factor 4 suppress the above acceleration (4, 5). It was subsequently showed that cultured cloned bovine macrovascular and rodent microvascular endothelial cells synthesize both anticoagulant HSPG (HSPGact) as well as nonanticoagulant HSPG (HSPGinact) (6-14 8). HSPGact bear glycosaminoglycan (GAG) chains that bind tightly to AT and accelerate T-AT complex generation (6-8).
The biosynthesis of HSPGact requires generation of a core protein, assembly of a linkage region of four neutral sugars on specific serine attachment sites of the core protein, elongation of a GAG backbone composed of alternating N-acetylglucosamine and glucuronic acid residues, and modification of this homogenous copolymer by partial N-deacetylation with coupled N-sulfation of glucosamine residues, partial epimerization of glucuronic acid to iduronic acid residues, partial 2-O-sulfation of uronic acid residues, and partial 6-O-sulfation and partial 3-O-sulfation of glucosamine residues (reviewed in 9)). This multienzyme pathway generates HSPGact with regions of defined structure that contain the primary AT binding domain sequence found in anticoagulant heparin: uronic acid→glucosamine (N-acetyl/N-sulfate) 6-O-sulfate→glucuronic acid→glucosamine N-sulfate 3-O-sulfate (6-O-sulfate)→iduronic acid 2-O-sulfate→glucosamine N-sulfate 6-O-sulfate (10-17). These reactions also produce HSPGinact with regions of varying monosaccharide sequence that lack the primary AT-binding domain. The structure-function relationships of the AT binding domain have been elucidated with heparin/heparan sulfate oligosaccharides in association with fast reaction kinetics and equilibrium binding assays. The 6-O-sulfate group on residue 2 and the 3-O-sulfate group on residue 4 function in a thermodynamically linked fashion to supply half of the binding energy for interaction with AT, and trigger a conformational event that accelerates neutralization of specific coagulation proteases (11, 12). The amino and ester sulfate groups at residues 5 and 6, as well as carboxyl groups at other sites, provide the other half of the binding energy for interaction with protease inhibitor (10, 11). Furthermore, monosaccharide sequences outside the primary AT binding domain are essential in facilitating inhibition of coagulation proteases other than factor Xa (18, 19).
During the past eight years, several biosynthetic enzymes that generate HSPGact and HSPGinact have been purified. These proteins include an N-acetylglucosamine/glucuronic acid copolymerase (20), N-deacetylase/N-sulfotransferases (NST-1 and NST-2) (21, 22), a glucuronic acid/iduronic acid epimerase (23), an iduronic acid/glucuronic acid 2-O-sulfotransferase (2-OST) (24), a glucosamine 6-O-sulfotransferase (6-OST) (25) and a glucosamine 3-O-sulfotransferase (3-OST) (26, 35). However, the only enzymes that have also been molecularly cloned are two structurally and functionally distinct isoforms of N-deacetylase/N-sulfotransferase (NST-1 from liver and NST-2 from mastocytoma) (27-31), and the 2-OST and epimerase. The above enzymes must function in a coordinated manner to produce the AT binding domain because the abundance of this sequence is much greater than predicted from a random assembly of constituents (32). The postulated regulatory mechanism must direct the biosynthetic enzymes to carry out the appropriate sequence of epimerization/sulfation reactions to generate the AT binding domain (33, 34).